Myocardial infarction (MI) remains one of the leading causes of morbidity and mortality in the developed world. For those fortunate to survive, the resultant ischemic myocardium can lead to significant morbidity including congestive heart failure, aneurysm formation, and repeat MIs. To date, scientist and clinicians have been unsuccessful in developing therapies for the significant regeneration of myocardium as stem cell-based strategies to restore muscle have shown limited effects in clinical trials. Therefore, a immediate need for basic research to elucidate novel approaches to heart regeneration remains. In our preliminary studies we have found evidence that the extracellular factor Neuregulin1 (Nrg1) is induced by cardiac injury, and that expression of this single factor in the absence of trauma is sufficient to stimulate robust myocardial hyperplasia in adult animals. The ability for Nrg1 to stimulate spontaneous creation of vascularized adult cardiac muscle holds great potential for addressing human cardiovascular disease. This proposal aims to exploit the robust regenerative capacity and Nrg1 response of the zebrafish to dissect and improve our understanding of Nrg1 signaling such that its therapeutic potential can be maximized. First, I will develop loss-of-function strains for Nrg1 and its receptor, Erbb4b, to investigate the specific mechanisms of the Nrg1 signaling pathway following ventricular resection in adult zebrafish. This work will be the first experiments to evaluate Nrg1 and its known receptors with conditional loss-of-function mutants in the setting of natural cardiac regeneration. To investigate downstream Nrg1 signaling mechanisms during cardiac regeneration I will employ cutting edge transgenic zebrafish lines to profile open chromatin states in cardiomyocytes (CMs). Specifically, these will be used to generate open chromatin and transcriptome profiles under increased endogenous Nrg1 signaling and in the setting of heart regeneration following injury. I will use these profiles to identify novel candidate Nrg1 downstream genes and pathways involved in heart proliferation and regeneration. Identified candidates genes will be assessed by in situ experiments along with the creation of knockout zebrafish lines. This improved understanding of Nrg1 downstream signaling will allow for the potential future targeting and activation of regeneration-specific pathways while minimizing the inherent systemic toxicity of Nrg1. If successful, the investigations in this proposal will dramatically further our understanding and the therapeutic potential of Nrg1-induced cardiac regeneration.